Determination of serum vitamins 25-OH-D2 and 25-OH-D3 with liquid chromatography–tandem mass spectrometry using atmospheric pressure chemical ionization or electrospray source and core-shell or sub-2 μm particle columns: A comparative study

ObjectiveDevelop a simple LC–MS–MS method for determination of 25-hydroxymetabolites of vitamin D2 and D3 in serum.Design and methods0.1 mL serum was mixed with 0.3 mL deproteinizing mixture containing two deuterated internal standards. Two LC–MS–MS systems were used: Waters Premier with an ESI source and a Hypersil Gold RP 18 50 × 2.1 mm column, and a Thermo Quantum, with an APCI source and a Kinetex RP 18 core-shell 50 × 2.1 mm column. Certified reference material 972 from NIST was used for method calibration. The samples were additionally analyzed with commercial HPLC procedure.ResultsBaseline separation of compounds was achieved in 6 min run time. Both systems have limits of detection of 0.5 nmol/L and recovery ranging from 86% to 112%. The measurement of 25-OH-D3 gave comparable results for all procedures; the results of 25-OH-D2 measurement obtained with the HPLC often differed from those obtained with LC–MS–MS methods.ConclusionsEstablished procedure performed well for ESI and APCI sources and totally porous and core-shell LC columns. The use of two internal standards enhanced the accuracy of the measurements.

► Fast method for determination of hydroxymetabolites of vitamins D2 and D3 with LC–MS–MS was developed.
► Both compounds were separated in 5 min time and individual deuterated internal standards were used.
► Two HPLC columns (UP and core shell) as well as two ionization sources (ESI and APCI) were used.